title: Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6.
abstract: The eukaryotic exosome exoribonuclease Rrp6 forms a complex with Rrp47 that functions in nuclear RNA quality control mechanisms, the degradation of cryptic unstable transcripts (CUTs), and in the 3' end maturation of stable RNAs. Stable expression of Rrp47 is dependent upon its interaction with the N-terminal domain of Rrp6 (Rrp6NT). To address the function of Rrp47 independently of Rrp6, we developed a DECOID (decreased expression of complexes by overexpression of interacting domains) strategy to resolve the Rrp6/Rrp47 complex in vivo and employed mpp6Δ and rex1Δ mutants that are synthetic lethal with loss-of-function rrp47 mutants. Strikingly, Rrp47 was able to function in mpp6Δ and rex1Δ mutants when separated from the catalytic and exosome-binding domains of Rrp6, whereas a truncated Rrp47 protein lacking its C-terminal region caused a block in cell growth. Northern analyses of the conditional mutants revealed a specific block in the 3' maturation of box C/D snoRNAs in the rex1 rrp47 mutant and widespread inhibition of Rrp6-mediated RNA surveillance processes in the mpp6 rrp47 mutant. In contrast, growth analyses and RNA northern blot hybridization analyses showed no effect on the rrp47Δ mutant upon overexpression of the Rrp6NT domain. These findings demonstrate that Rrp47 and Rrp6 have resolvable functions in Rrp6-mediated RNA surveillance and processing pathways. In addition, this study reveals a redundant requirement for Rrp6 or Rex1 in snoRNA maturation and demonstrates the effective use of the DECOID strategy for the resolution and functional analysis of protein complexes.
authors.forename: William
authors.initials: W
authors.lastname: Garland
authors.forename: Monika
authors.initials: M
authors.lastname: Feigenbutz
authors.forename: Martin
authors.initials: M
authors.lastname: Turner
authors.forename: Phil
authors.initials: P
authors.lastname: Mitchell
mesh_terms: D001667:Binding, Competitive; D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D015870:Gene Expression; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012331:RNA, Fungal / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017384:Sequence Deletion
chemical_list: D004268:DNA-Binding Proteins; C494789:LRP1 protein, S cerevisiae; C531213:Mpp6 protein, S cerevisiae; D009687:Nuclear Proteins; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C529500:Rex1 exonuclease, S cerevisiae; C112610:RRP6 protein, S cerevisiae
keywords: RNA processing; RNA surveillance; exosome; protein overexpression; synthetic lethality; yeast